New cobalamins



D. PERLMAN NEW COBALAMINS Filed Jan. 22, 1957 Feb. 3, 1959 NH co-cH cH;

A um-co-cg HC um-to-cm-cn co-cn CH 2 2 United States Patent pub NEW 'COBALAMINS David Perlman, Princeton, N. L, assignor to Olin Mathie son Chemical Corporation, New York, N. Y., a corporation of Virginia This invention relates to new cobalamins and, more particularly, to (phenazin-9,10-diyl)-hydroxo-cobalamins and salts thereof [the nomenclature herein is based on Bernhauer et al., Angew. Chemie, 66, 776 (1954)]; and to a biosynthetic method of producing these new cobalamins employing suitable precursors.

The new compounds of this invention may be represented by the structural formula in the appended drawing, wherein X is an anion, for example an hydroxy radical or the anion of an acid, preferably a pharmacologically-acceptable acid and Y is a (phenazin-9,10- diyl) group. Examples of suitable anions are the anions of the mineral acids (e. g., chloride, bromide, sulfate, phosphate, nitrite and nitrate), cyanide, cyanate etc.

The phenazin-9,l0-diyl group, Y, may be unsubstituted or may contain one or more R-substituents (preferably no more than two) such as alkyl, alkoxy, hydroxy, halo, amino, nitro or acetamido group; and it is attached to the X-cobalamin through both nuclear nitrogen atoms. Thus the compounds of this invention, termed (phenazin-9,10-diyl)-X-cobalamins, include for example: (2-methylphenazin-9, l0-diyl) -cyano-cobalamin; [2- (apt, vgy-tetramethylbutyl)phenazin 9,10 diyll-hydroxo-cobalamin; (3-ethylphenazin-9,lO-diyl)-cyano-cobalamin; (3,6-diaminophenazin 9,10 diyl)-hydroxo cobalamin; (3 ,6-diaminophenazin-9, 1 O-diyl) -cyano-cobalamin; (3- amino-6-dimethylaminophenazin 9,10 diyl)-cyano-cobalamin; (6-aminophenazin-9,10-diyl)-cyano-cobalamin; (1-acetamido-3-ethoxyphenazin 9,10 diyl)-chloro-cobalamin; 1,6-dimethoxyphenazin-9,10-diyl)-chloro-cobalamin; (1,6-dichlorophenazin-9, 1 O-diyl) -sulfato-cobal arnin; (3-amino-2-hydroxyphenazin-9,l0-diyl)-hydroxycobalamin; 1-amino-2-hydroxyphenazin-9, l 0-diyl) -hydroxy-cobalamin; (2-nitro-phenazin-9, l0-diyl -chloro-cobalamin; (2-bromo-phenazin-9, 1 O-diyl) -hydroxo-cobalamm.

The compounds of this invention are biologically active, and hence can be used in lieu of vitamin B (5,6-dimethylbenzimidazole-cyano-cobalamin) in promoting growth of chicks. For this purpose, they are administered in the same manner as vitamin B (e. g., as supplements to chick feeds or by injection), the respective dose of the particular (phenaZin-9,10-diyl)-X-cobalamin depending on its potency relative to vitamin B To prepare the (phenazin9,10-diyl)-X-cobalamins of this invention a Propionibacterium (such as Propionibacterium rubrum, Propionibacterium zhoenii, Propionibacterium zeae, Propionibacterium peterssonii, Propionibacterium pemosaceum, Propionibacterium jensenii and preferably Propio nibacterium arabinosum) is grown under substantially anaerobic conditions in a suitable nutrient medium containing a selected precursor.

Precursors utilizable in the practice of this invention include a phenazine which is unsubstituted or which contains one or more R substituents, as explained hereinbefore (which may be the same or different) or the salts (acid addition or quaternary salt thereof) and other derivatives based on the functional amino groups of the .30 g; autolyzed yeast, 20 g.; Co(NO -6H O, 0.01 g.;

phenazine. Examples of suitable precursors include phenazine; amino, nitro or acetamido-substituted phenazine (such as: Z-aminophenazine; Z-nitrophenazine; l-acetamido-3-methoxy-phenazine); an hydroxy-substituted phenazine (such as: 1-amino-2-phenazinol; 3-amino-2- phenazinol; 2-phenazi11ol); and halogen-substituted phenazine (such as, Z-bromophenazine; 2-chlorophenazine). The sources of carbon, nitrogen and cobalt, utilizable as media components in this process, are those normally employed in the preparation of vitamin B The resultant (phenazin-9,l0-diyl)-hydroxo-cobalamin is then converted, in situ, to (phenazin-9,l0-diyl)-cyano-cobalamin by treatment of the culture or separated cells (preferably the latter) withpotassium cyanide or other cyanide containing salt, and the cyano-cobalamin is isolated as more fully detailed in Example 1. The resultant cyano-cobalamin can then be converted to hydroxo-cobalamin and thence to other salts of (phenazin-9,l-0-diyl)-cobalamin by methods known in the art [see Kaczka et al., Jour. Amer. Chem. Soc, 73,3569 (1951)] to yield products which are also biologically active; alternatively, the (phenazin-9,10-diyl)-hydroxo-cobalamin initially obtained may be used to prepare the other cobalamin salts.

. The following examples illustrate the invention (all temperatures being in centigrade):

EXAMPLE 1 (Phenazin-9,10-diyl)-cyan0-c0balamin (X:CN)

30 liters of a medium containing (per liter): glucose,

tap Water, 1 liter; are placed in a stainless steel fermentation unit of 38 liter capacity, heated at 121 for 30 minutes, and cooled to 30. About 2,000 ml. of a slurry of CaCO (containing 600 g. of CaCO sterilized by autoclaving, is then added together with 1 liter of Propionibacterium ambinosum (American Type Culture Collection catalogue Number 4965, Washington, D. C.) culture grown on this medium for 72 hours in flasks shaken on a reciprocating shaker (120 l-inch strokes per minute), located in a 30 constant temperature room. A sterile alcoholic solution of phenazine (sterilized by filtration through a fritted glass filter) containing 1 mg. per ml. is then added so that the final concentration of the phenazine is 5 mg. per liter (this supplementation being repeated at 24-hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 R. P. M. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is 30 g. per liter. After 5 days of incubation, the pH of the mixture is about 5.3. The fermented medium is then passed through a Sharples Super Centrifuge, or the cells containing (phenazin-9,10-diyl)-hydroxo-cobalamin are separated 'by filtration through a bed of filter-aid (Celite) The collected cells are resuspended in 3 liters of propanol-water (80:20) containing 0.1 g./liter of potassium cyanide, and the suspension is heated at 80-90" for 20 minutes. After filtration, the filtrate is concentrated to about 750 ml. The pH is adjusted with 10 N sodium hydroxide to about 7.5, and 25 g. of activated carbon (Darco G) is added and the mixture stirred. The activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (:35 v./v.), containing 0.1 g./liter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three 400 ml. volumes of phenol-benzene parts 88% phenol-30 parts benzene). The non-aqueous layer is separated, pooled and diluted with benzene so that the ratio of benzene to phenol is 15:1. The solucitric acid, 1.2 g.; (NH PO 0.4

tion is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concentrated to about 50 ml. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the rich-water is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous layer is reextracted with one-tenth its volume of benzyl alcohol. The rich benzyl alcohol extracts are combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column is washed with 1 part of methanol and 2 parts of acetone. The rich material is eluted with methanol and the eluate dried under vacuum. The residue is dissolved in a minimum of water and acetone is added until slightly turbid. On standing for several days (phenazin-9,10-diyl)-cyano-cobalamin in the form of red needlelike crystals is recovered. Spectroscopic examination shows maxima at 361, 520 and 550 my.

The product (phenazin-9,10-diyl)-cyano-cobalamin is readily obtained, under the same conditions, using Propionibacterimn pentosaceum (ATCC Number 4875) or Propionibacterium peterssonnii (ATCC Number 4870) in place of Propionibacterium arabinosum in Example 1.

To show the homogeneity and activity of the (phenazin- 9,10-diyl)-cyano-cobalamin of this invention, the following tests were conducted. For these tests the cyano-cobalamin was dissolved in water at a concentration of 100 micrograms of cyano-cobalamin per ml. of water:

TEST I The solution of (phenazin-9,10-diyl)-cyano-cobalamin is dried on a filter paper strip of Whatman 3mm. paper in parallel with samples of 5,6-dimethyl-benzimidazolecyano-cobalarnin, adenine-cyano-cobalamin, 2-methyl- 'adenine-cyano-cobalamin, and Fords factor B [Ford et al., Biochem. Jour., 59, 86 (1955)]. The sheet is placed in an ionophoresis apparatus [similar to that described by l-lolclsworth in Nature, 171, 148 (1953)], and the paper is impregnated with a solution of 0.5 In. acetic acid containing 0.02% KCN (W./v.). A potential of about 280 volts is applied for about 17 hours. The sheet is removed and dried. When dry (and free from odor of acetic acid), it is applied for minutes to the surface of an agar plate seeded with a suspension of a vitamin B -requiring strain of Escherichia coli (ATCC 11105). [The agar medium contains grams/ liter): sucrose, g.; g.; KCl, 0.08 g.; MgCl -6H O, 0.418 g.; MnCl -4H O, 0.036 g.;

FeCl 6H O, 0.023 g.

ZnCl 0.021 g.; CoCl -6H O, 0.04 g.; agar, 15 g.; triphenyl tetrazolium chloride. 1 g.]. After 18 hours incubation at 37, the agar plate is observed. The positions of zones of growth of the bacteria (noted as red zones due to the reduction of the tetrazolium dye to the colored formazan) are noted in relation to the location on the paper strip where the samples were applied. When the samples from the fermentation supplemented with the phenazine are analyzed in this Way, zones of growth parallel to those obtained when 5,6-dimethylbenzimidazole-cyano-cobalamin (neutral ionophoretically), adenine-cyano-cobalamin and factor B are found, with the largest zone in the neutral area.

TEST II Another aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatman No. 1 filter paper parallel to spots of known cobalamins. The chromatogram is developed by the descending method using a solvent mixture containing: 77 m1. of sec-butanol, 23 ml. H O, 0.25 ml. KCN solution (5 gms./ml.) and 100 mg. KClO for 24 hours (at 25). The strip is dried and applied to the seeded agar plate as above. After incubation, the zones of growth, representing the presence of vitamins of the B group (measured with reference to the movement of 5,6-dimethylbenzimidazolecyano-cobalamin), were 0.5, with smaller zones of growth corresponding to 0.38 (adenine-cyano-cobalamin) and 1.45 (factor B).

TEST III Another aliquot of the solution is applied to a spot about 3 inches from the end of strip of Whatman No. 4 filter paper parallel to spotsof known cobalamins. The chromatogram is developed 'by the descending method a solvent mixture containing: sec.-butanol, 100 ml.; r1 0, 50 ml; KCN [5% solution (w./v.)l, 0.25 ml; NI-hOI-I (concentrated), 1.0 ml. After 17 hours development (at C.), the strips are dried and plated on seeded agar plates as above. Zones of growth indicating the presence of new substance were obtained with a mobility of 0.35 that of the 5,6-dimethylbenzimidazolecyano-cobalamin are found, with smaller zones in the area of mobility about 0.65 and 1.25 that of the 5,6-dimethylbenzimidazole-cyano-cob'alamin (corresponding to adenine-cyano-cobalamin and factor B respectively).

TEST IV Another aliquot is assayed for the presence of substances stimulating the growth of Lactobacillus leichmannii (ATCC 7830) using as standard 5,6-dimethyloenzimidazole-cyano-cobalamin and the method in the U. S. Pharmacopoeia (15th edition). A value of about 0.2 mg. per liter is obtained. A second aliquot is assayed by the method of Ford and Porter [Brit. J. Nutrition, 7, 326 (1953)], using the growth response of Ochromonas malhamensis, and the 5,6dimethylbenzimidazole-cyanocobalamin as standard; a value of about 0.16 mg. per liter is obtained. When an aliquot from an unsupplemented fermentation is analyzed by these procedures, no growth stimulation of the Ochromonas culture is found (less than 0.05 mg. per liter), and no evidence for production of ionophoretic'ally neutral cobalamins is obtained.

TEST V Another aliquot is assayed for the presence of substances stimulating the growth of chicks, using the procedure described by Lillie et al. [Poultry Science, 33, 686 (1945)l with replacement of B'y 500 and bone meal with riboflavin and dicalcium phosphate. After 4 weeks growth, the chicks grown on the ration supplemented with the phenazin-9,IO-diyI-cyano-cobalamin were approximately equal in weight to those grown on the ration supplemented with an equal weight (based on the Lactobacillus leichmannii bioassay) of vitamin B EXAMPLE 2 (Phenazin 9,1 O-diyl) -hydrox0-c0balamin (X=OH) lia EXAMPLE 3 (Phenazine-9,10-diyl) -chloro-c0balamin (X=Cl) 10 mg. of (phenazin-9,IO-diyl)-hydroxo-cobalamin is dissolved in 3 ml. of water and the solution adjusted to pH 4 with 0.1 N hydrochloric acid. The solution is then diluted with 10 ml. of acetone, and after a short time the chloro-cobalamin begins to crystallize in the form of dark red needles. The crystals are separated by centrifuging, washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium.

(2-nitr0phenazin-9,10-diyl) -cyan-c0balamin (X=CN) Aliquots of the autolyzed yeast-glucose- CO z water medium described in Example 1 are placed in Erlenmeyer flasks (500 ml. per filter flask is a suitable quantity). The flasks are plugged with non-absorbent cotton and autoclaved for 20 minutes at 121 C. After cooling to room temperature, 10 grams of calcium carbonate powder (previously sterilized by autoclaving at 120 C. for 30 minutes and heating in a 150 C. oven for 1 hour) is added. The flasks are inoculated with 10 ml. of a 72 hour culture of Propionibacterium arabinosum (American Type Culture Collection catalog Number 4965) and placed on a reciprocating shaker (120 l-inch strokes per minute) located in an incubator maintained at 30 C. Two ml. of a sterile alcoholic solution of 2- nitrophenazine is added so that the final concentration of the phenazine is mg. per liter. This addition is repeated 6 times at 24 hour intervals. Twenty ml. of a solution containing 50 grams of glucose per 100 ml. is added after 3 days incubation. After 6 days incubation on the shaker the fermented medium is heated at 9095 C. for 20 minutes. Ten ml. of a KCN solution (5 g./ 100 ml.) are added per liter of fermented medium and the solids separated by centrifugation. The supernatant liquid (pH approx. 5.6) is extracted thrice with /2 its volume of a phenol-benzene solution (70 parts 88% liquified phenol-30 parts benzene). The benzene extracts are pooled and diluted with an equal volume of n-butanol. A quantity of water equivalent to the volume of the phenol-benzene-butanol solution is added and the mixture shaken vigorously and centrifuged. The red colored aqueous layer is separated and analyzed by the tests described in Example 1.

When analyzed by the ionophoretic method (pl-I 2.5) a cobalamin with ionophoretically neutral properties is found. When analyzed by the filter paper chromatographic method using the solvent system sec.-butanol, 100; water, 50; 5% KCN, 0.25 ml. and glacial acetic acid, 1.0 ml. a cobalamin with mobility of 0.24 that of 5,6- dimethylbenzimidazole-cyano-cobalamin is found. The solution containing (2-nitrophenazin-9,lO-diyl)-cyanocobalamin also stimulates the growth of a cobalaminrequiring culture of Ochromonas malhamensis.

EXAMPLE 5 (Z-aminophenazine-9,I0-diyl)-cyan0-c0balamin The procedure and method of testing the aqueous extract described in Example 4 is repeated except that a molar equivalent of 2-aminophenazine is used instead of 2-nitrophenazine. The cobalamin is found to be ionophoretically neutral (when the pH of the system is 2.5). When studied by filter paper chromatography using the system containing sec.-butanol, water, KCN, and glacial acetic acid, the new cobalamin has a mobility of 0.4-5 relative to that of 5,6-dimethylbenzimidazole-cyanocobalamin. If the system containing sec.-butanol, water, KCN, and KClO is used, the mobility of new cobalamin is 0.28 of that observed when 5,6-dimethylbenzimidazole cyano-cobalamin is added. The new cobalamin stimulates the growth of Ochromonas malhamensis.

EXAMPLE 6 (I -aminc-2-hydroxy-phenazin-9,1 O-diyl -cyano-c0balam in The procedure and method of testing the aqueous extract described in Example 4 is repeated except that a molar equivalent of 1-amino-2-phenazinol is used in place of Z-nitrophenazine.

The cobalamin formed is found to be ionophoretically neutral (when the pH of the system is 2.5); and in the filter paper chromatographic system using sec.-butanol, water, KCN, and acetic acid for developing solvent, the (1-amino-2-hydroxy-phenazin-9,lO-diyl) cyano cobalamin has a mobility 0.21 that of 5,6-dimethylbenzimidazole-cyano-cobalamin. The new cobalamin stimulates the growth of Ochromonas malhamensis.

EXAMPLE 7 (1-acetamid0-3-meth0xy-phenazin-9,1O-diyl) -cyan0- cobalamin The procedure and method of testing the aqueous extract described in Example 4 is repeated except that a molar equivalent of 1-acetamido-3-methoxy-phenazine is used instead of the 2-nitrophenazine. The new cobala min, (l-acetamido 3 methoxy phenazine 9,10 diyl)- cyano-cobalamin is found to be ionophoretically neutral (when the pH of the system is 2.5); and the mobility of the new cobalamin in the filter paper chromatographic system using sec.-butanol, water, KCN, and KClO is 0.13 that of 5,6 dimethylbenzimidazole cyano cobalamin. The new cobalamin stimulates the growth of Ochromonas malhamensis.

' EXAMPLE 8 (2-hydroxy-phenazin-9,I O-diyl) -cyano-c0balamin As an alternative to the procedure described in Example 4 the culture of Propionibacterium arabinosum may be grown on the autolyzed yeast-glucose- Co (N0 2 6H OCaCO medium for 2 days (without the inclusion in the medium of phenazine or derivatives of phenazine). At the end of this period the solids (cells, debris, and CaCO are separated from the liquid by centrifugation and resuspended in a volume of distilled water so that the cell concentration is approximately the same as before centrifugation. The suspension is well agitated and the solids collected again by centrifugation. These solids are then resuspended in distilled water equivalent to half the volume before centrifugation. Aliquots of this suspension are distributed into flasks (20 ml. per 125 ml. Erlenmeyer flask is a convenient volume) and 1 m1. of an aqueous solution of 2-phenazinol is added to one of a group so that the concentration of phenazinol is approximately 10 mg. per liter. These flasks are placed on a reciprocating shaker (120 l-inch strokes per minute) located in a room maintained at 30 C.

After 18 hours shaking 0.5 ml. of 5% KCN is added and the contents of the flasks are heated at to C. for 30 minutes in a boiling Water bath. The solids are collected by centrifugation and the supernatant liquid extracted with the phenol-benzene solution as in Example 4. The benzene extracts are pooled, an equal volume of n-butanol is added, and the mixture extracted with its volume of water. The aqueous layer is separated and analyzed by the tests described in Example 1.

When analyzed by the ionophoretic method (pH 2.5), the cobalamin formed, (2-hydroxy-phenazin-9,l0-diyl)- cyano-cobalamin, is ionophoretically neutral. When analyzed by the filter paper chromatographic method using the sec.-butanol, water, KCN and acetic acid system the cobalamin has a mobility about 0.8 that of 5,6-dimethylbenzimidazole-cyano-cobalamin. (When samples are taken from cell suspensions which have not been supplemented with any phenazines, only adenine-cyanocobalamin appears to be present according to the tests of Example 1.).

In an analogous manner, by substituting a molar equivalent of 3-amino-2-phenazinol or 2-bromophenazine in place of 2-phenazinol in Example 8, the corresponding cobalamins, namely (3-amino-2rhydroXy-phenazin-9,10- diyl)-cyano-cobalamin and (2 bromo phenazin 9,10- diyl)-cyano-cobalamin are formed.

In the practice of this invention, the isolated new cobalamin (or a fermentation residue or concentrate containing the new cobalamin, such as the dried fermentation medium) can be employed advantageously as an animal feed supplement.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

I claim:

1. A compound of the class consisting of (phenazin- 9,10-diyl)-hydroxo-cobalamin and the cyanide salts thereof.

2. A Y X-cobalamin wherein X is a member of the class consisting of hydroxo and cyano, and Y is a phenazin-9,10-diyl group of the general formula wherein each R is a member of the class consisting of hydrogen, alkyl, alkoxy, hydroxy, halo, amino, nitro and acetamido, the Y group being attached to the X-cobalamin through both nuclear nitrogen atoms.

3. (2-arnino-phenazin-9,IO-diyl)-cyano-cobalamin.

4. (Phenazin-9,10-diyl)-cyano-cobalamin.

5. (1-amino-2-hydr0Xy-phenazin-9,10 diyl) cyano cobalarnin.

6. 1-acetamido-3-methoxy-phenazin-9,10-diyl)-cyanocobalarnin.

7. (2-hydroXy-phenazin-9,10-diyl)-cyano-cobalamin.

References Cited in the file of this patent UNITED STATES PATENTS 2,595,499 Wood et al. May 6, 1952 2,650,896 McDaniel et al. Sept. 1, 1953 2,719,843 Davoll et a1 Oct. 4, 1955 2,728,763 Mamalis et al. Dec. 27, 1955 2,763,642 Porter et a1 Sept. 18, 1956 OTHER REFERENCES Bernhauer et al.: Angewandte Chemie. 66:24, Dec. 21, 1954, pages 776-780 relied on. 

1. COMPOUND OF THE CLASS CONSISTING OF (PHENAZIN9,10 DIYL) -HYDROXO-CABALAMIN AND THE CYANIDE SALTS THEREOF. 